Estrogens are responsible for the growth, development and maintenance of many reproductive cells. The physiological effects of these hormones are mediated by a ligand-inducible nuclear transcription factor, the estrogen receptor (ER). In the classical pathway of steroid hormone action, 17β-estradiol binds to the ligand binding domain (LBD) of an estrogen receptor and induces homodimerization, which then binds to a specific regulatory sequence of promoters of ER target genes, the estrogen response elements (ERE). The binding of hormones and a variety of other chemicals to the LBD of ER leads to a series of downstream molecular events. This includes the activation or repression of many downstream target genes through direct interaction with the transcription machinery.
Abnormal levels of estrogen have been linked with many diseases and disorders including cancer. The deficiency in the level of estrogen in post menopausal women can lead to reduced bone densities. Similarly, the presence of excess hormones has been reported to induce the development of different types of cancers including breast cancer. Most of these cancers respond to hormonal therapy (anti-estrogens) via the estrogen receptor. Hence, estrogen receptors are a major cellular therapeutic target.
The ER-LBD is folded into a three-layered, anti-parallel, α-helical sandwich composed of a central core layer of three helices that includes H5/6, H9, and H10. This is in turn sandwiched between two additional layers of helices (H1-4 and H7, H8, H11). This helical arrangement creates a “wedge shaped” molecular scaffold that maintains a sizeable ligand binding property at the narrower end of the domain. The remaining secondary structural elements, a small two-stranded, anti-parallel β-sheet (S1 and S2) and an α-helical H12, are located at this ligand binding portion of the molecule and flank the three-layered motif. The helix 12 (H12) is mainly located in the pocket of the ligand binding region. Therefore, it is a key element of the receptor in developing conformational modifications in response to various ligands. The crystal structures of the LBD complexed with 17β-estradiol and Raloxifene show that although both ligands bind at the same site within the core of the LBD, each of these ligands induces a different conformational change on H12. In addition, the binding of ligands to the ligand-binding domain of ERα causes a conformational shift of helix 12 into an adjacent co-activator site that either prevents or enhances ERα from binding to a co-activator (NR box peptide), which would then activate a specific DNA sequence, the estrogen response element (ERE). This process controls many genes that are responsible for cell growth. Hence, helix 12 is one of the major portions of ER that plays a critical role in the ligand-induced proliferative effect of cells, and it is therefore important to develop an assay based on the movement of helix 12 in response to different ligands.
To date, several assays have been developed for screening ER ligands by using either purified ER protein or ER from cell lysates. Very few fluorescence resonance energy transfer (FRET) based assays have been used to study ER ligands in intact cells. FRET measures either ligand induced conformational changes while using the full length ER or the recruitment of co-activator peptides (LXXLL) by ER in response to ligand binding. For example, FRET measuring ligand induced conformational change with full length ER was used to study the phosphorylation mediated arrest induced by tamoxifen in breast cancer cells. FRET is a semi-quantitative assay and does not currently translate to imaging living animals. Some assays have been designed to study the effects of chemical agonists and antagonists of ER through their downstream target gene activations.